1. Cut some mycelial plugs (like 5) from a 10 -14 days old PDA culture.
2. Inoculate with the agar plugs a flask with 100 ml of Fries medium 48 hours with shaking at 30 C. I use a 100 mL volume culture in a 250 mL Erlenmyer flask and shake 250rpm .
3. Filtrate culture in funnel with miracloth. Keep mycelium and spores. Trash agar plugs.
4. Blend the culture filtrated with a 100 ml of new Fries medium. Use a sterile blender.
5. transfer to 1 L sterile flask . Complete to 400 ml of Fries medium
6. Incubate 24 hs at 30C at 250 rpm (400 ml f Fries media in a 1 L flask)
7. Filter through a funnel with sterile paper with vacum presure.
8. Divide into aliquots , cool in liquid N2 for 1 min and freeze at -80C. I usually use 100 mg in 1.5 ml tubes or 1 gr in a 50 ml tubes aliquots. Also it can be crash in the mortle first and after that storage.
Material to be ready before start
Baked: clean wipe with RNA free and bake 12 hs at 170 C.(second floor)
Mortle and plestle , ceramic funnel, spatulas (2)
Sterile Flask 250 ml and 1 L
Sterile Funnel with Miracloth
Sterile whatman paper (size: ceramic funnel)
Metal Blender flask ( kennerley lab)